Enhancers that transcriptionally activate promoters from several kilobases away are vital for expression of many genes during metazoan development. The Chip protein was discovered in a genetic screen for factors that support activation of the Drosophila cut gene by a distant enhancer. Chip, also found in humans, facilitates activation of many Drosophila genes, including eve, by remote enhancers. The primary goal of this proposal is to determine how Chip facilitates activation of eve. Chip interacts with homeoproteins such as Bicoid and increases Bicoid activity in vivo. The principal hypothesis is that Chip increases cooperative binding of Bicoid and other homeoproteins to eve enhancers. Another important idea is that Chip also helps homeoproteins bind between the eve enhancers and promoter and thereby facilitates enhancer-promoter communication. Gypsy transposon insertions can block communication between enhancers and their target promoters in many genes, including eve and cut. Chip is a genetic target of the gypsy insulator at the cut locus, and interacts with proteins that bind gypsy, Su(Hw) and Mod(mdg4)-67.2, implying that Chip is also a molecular target of the insulator. The proposed work has four aims: (1) to determine how Chip promotes Bicoid activity using in vitro DNA-binding assays and in vivo gene expression experiments; (2) to test if Chip interacts directly with eve in vivo, and where in eve it binds using chromatin immunoprecipitation assays; (3) to determine if a remote eve enhancer that is Chip-dependent comes physically close to the eve promoter during activation using an in vivo site-specific recombination assay; and (4) to define how the Su(Hw) and Mod(mdg4)-67.2 insulator proteins interact with each other and Chip using in vitro protein interaction assays, and in vivo genetic experiments. These studies will increase understanding of long-range gene activation and illuminate fundamental mechanisms underlying some human genetic diseases.